Insect controlling agent from strains of Bacillus thuringiensis var. Kurstaki

ABSTRACT

A method for protection of a plant from damage caused by an insect pest of the order Lepidoptera and Diptera is disclosed. An insect controlling agent having an active component of an insecticidal crystal toxin produced by Bacillus thuringiensis var. kurstaki No. 145 FERM BP-3905, No. 161 FERM BP-3906, and No. 116 FERM BP-3907 is used in the method for protection of a plant. The insecticidal crystal toxin has a set of molecular weights as determined by 10% SDS-polyacrylamide gel electrophoresis of 125,000 daltons and 60,000 daltons when produced by No. 145, 130,000 daltons and 60,000 daltons when produced by No. 161, and 130,000 daltons and 60,000 daltons when produced by No. 116.

This is a divisional application Ser. No. 07/911,570 filed on Jul. 9,1992, now U.S. Pat. No. 5,266,483.

FIELD OF THE INVENTION

This invention relates to novel bacteria strains, particularly to novelBacillus thuringiensis (which will be referred to hereinafter as "Bt")strains and application thereof.

The invention relates to the novel strains which produce an insecticidalcrystal toxin. The toxin can control lepidopterous insect pests moreeffectively than commercially available agents. The lepidopterous insectpests cause serious damages to plants in a farming field, non-farmingfield and forest.

The invention further relates to an agent for controlling thelepidopterous insect pests, which comprises the novel strains or crystaltoxin produced thereby.

BACKGROUND

Bt is a gram-positive bacilliform bacterium which produces a crystalprotein at a sporulation stage in the end of a logarithmic growth phase.When an insect orally takes the crystal protein into a gastrointestinaltract, the crystal protein will be subjected to alkaline and enzymaticdegradations in a digestive juice so as to show an insecticidal activitywhich causes an intestinal and systemic paresis. The crystal protein istherefore referred to as "δ-endotoxin" (Heimpel, A. M.; Ann. Rev.,Entomology 12, 287-322, 1967).

The crystal protein produced by Bt is generally of a form such as adiamond-shaped, bipyramidal and rhomboidal one. The crystal protein isformed with an endospore in a sporangium and released with it from thesporangium (Hannay, C. L.; Nature 172, 1004, 1953).

Bt has been classified on the basis of H-antigen according to theproposal by De Barjac and Bonefoi (Entomophaga 7, 5-31, 1962), andreported to have 24 subspecies (33 strains) including serotypes 1 to 23and wuhanensis which has no H-antigen (Toshihiko Iizuka, Chemistry andBiology 27, 287-302, 1989). However, it was found that some differentkinds of strains were present in the same serotype (Krywienczyk, J., etal; J. Invertebr. Pathol.; 31, 372-375, 1978, Iizuka, T., et al; J.Sericult. Sci. Japan; 50, 120-133, 1981).

The crystal protein produced by Bt is generally known to selectivelyshow the insecticidal activity to the lepidopterous insects. Recently,subspecies have been found, which produce an irregular and cuboidalcrystal protein beside a bipyramidal one (Yoshio Akiba; Jpn. J. Appl.Entomol. Zool., 30, 99-105, 1986, Ohba, M., et al; J. Invertebr. Pathol.38, 307-309, 1981). Among them, there were reported strains which canshow a strong insecticidal activity against a leaf beetle or a larva ofa mosquito (Hall, I. M., et al; Mosquito news 37, No.2, 1977).

A crystal protein gene (CP gene) of Bt is usually encoded by a plural ofsomatic plasmid DNAs. The CP gene has been already cloned and itsnucleotide sequence has been also determined in some subspecies(Toshihiko Iizuka, Chemistry and Biology 27, 287-302, 1989).

Further, many researches have been intensively conducted on introductionof such cloned CP genes into a plant body and some of them were reportedto succeed, for example, in the case of tobacco (Vaeck, M., et al;Nature 328, 33-37, 1987). The CP gene coding for a crystal protein whichshows a stronger insecticidal activity is now sincerely desired.

The lepidopterous insect pests having taken the crystal toxin producedby Bt will stop their feeding behavior several hours later and never doany harm to plants. Almost species of the lepidopterous insect pestswill die of intoxication by the crystal toxin about 24 to 72 hourslater. The intoxication will be sometimes accompanied by sepsis inducedby the presence of spores. Thus, a main reason of the death is due tothe crystal toxin which will function only after its dissolution in abowel of the larva.

Due to such functional mechanism of the crystal toxic protein as well asthe fact that the crystal toxin is an easily-degradable polypeptide, itis known that the crystal toxin does no damage to human, pet animals,birds, fishes and plants.

Accordingly, Bt or the crystal toxic protein produced thereby is verypotential as a microbial pesticide which does not pollute an environment(BT agent), especially as an insecticide against the lepidopterousinsect pests. One or more strains of Bt have been actually used for along time as the insecticide in agriculture.

The strain of Bt which is most generally used as a commercial product isBt var. kurstaki HD-1, which will be referred to hereinafter as "HD-1."

SUMMARY OF THE INVENTION

The present inventors have found novel strains of Bt. The new strains ofthe present invention are definitely distinguished from HD-1 by the factthat they show an improved insecticidal activity against a series ofdipterous and lepidopterous insect pests but they show only a lowinsecticidal activity against a beneficial lepidoptera, Bombyx mori.

Accordingly, one object of the present invention is to provide novelstrains of Bt var. Kurstaki, No.145, No.161 and No.116, which have beenoriginally deposited at Fermentation Research Institute Agency ofIndustrial Science and Technology on Jun. 4, 1991 with Accession NumbersFERM P-12289, FERM P-12291 and FERM P-12290, respectively. These strainshave been transferred from the original deposit to the deposit underBudapest Treaty conditions on Jun. 29, 1992 with new Accession NumbersFERM BP-3905, FERM BP-3906 and FERM BP-3907, respectively.

Another object of the present invention is to provide a novelinsecticide composition or insect pest-controlling agent, whichcomprises as a main component an insecticidal crystal toxin produced bythe novel strains No.145, No.116 and No.161 according to the presentinvention.

Yet another object of the present invention is to provide a method forthe protection of a plant from damage caused by an insect pest,comprising feeding the insect pest with the insecticidal crystal toxinproduced by the novel strains No.145, No.116 and No.161 according to thepresent invention or treating a plant with an insecticidally effectiveamount of the same toxin prior to or during suffering from the damage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a pattern on SDS-polyacrylamide gel electrophoresis of theproteins which were obtained by a trypsin treatment of the alkalinesoluble protein of the strain No.116 (lane 1), No.145 (lane 2), No.161(lane 3) and HD-1 (lane 4), respectively. Lane 5 is a pattern of trypsinand lane 6 shows migration markers.

DETAILED DESCRIPTION OF THE INVENTION

The above three novel strains according to the present invention wereisolated from soil in Hokkaido area and identified to have the followingfeatures.

Features of the strain No.145:

Colony Formation: A large colony which is typical for Bt, the surface ofwhich looks dark and dull.

Cell morphology: A typical one for Bt.

Serotype of H-antigen: 3a3b3c, kurstaki.

Intracellular component: A sporulating cell produces a diamond-shapedcrystal which sometimes contains a small cuboidal crystal.

Plasmid: A pattern of a plasmid of the strain on an agaroseelectrophoresis is clearly distinguished from that of the known strainssuch as Bt var. kurstaki HD-73 (which will be hereinafter referred to as"HD-73") and Bt var. kurstaki HD-263 (which will be hereinafter referredto as "HD-263"). Alkaline-soluble protein: The strain has the proteinswhich run to about 125,000 dalton and about 60,000 dalton on theelectrophoresis.

Activity: The strain has an insecticidal activity which is from 5 to 100times higher than the known HD-1 and commercially available Bt agentsagainst all the lepidopterous insect pests tested, but has theinsecticidal activity which is about 20 times lower than the known HD-1and commercially available Bt agents against a beneficial lepidoptera,Bombyx mori. The strain also shows a stronger insecticidal activity thanHD-1 against a dipterous insect pest, mosquito.

Features of the strain No.116:

Colony Formation: A large colony which is typical for Bt, the surface ofwhich looks dark and dull.

Cell morphology: A typical one for Bt.

Serotype of H-antigen: 3a3b3c, kurstaki.

Intracellular component: A sporulating cell produces a diamond-shapedcrystal which sometimes contains a small cuboidal crystal.

Plasmid: A pattern of a plasmid of the strain on an agaroseelectrophoresis is clearly distinguished from that of the known strainssuch as HD-1, HD-73 and HD-263. Alkaline-soluble protein: The strain hasthe proteins which run to about 130,000 dalton and about 60,000 daltonon the electrophoresis.

Activity: The strain has an insecticidal activity which is from 5 to 80times higher than the known HD-1 and the commercially available Btagents against all the lepidopterous insect pests tested. The strainalso shows a similar insecticidal activity to Bt var. israelensis (whichwill be hereinafter referred to as "israelensis") against dipterousinsect pest, mosquito. However, it has a lower insecticidal activitythan the known HD-1 and the commercially available Bt agents against abeneficial lepidoptera, Bombyx mori.

Features of the strain No.161:

Colony Formation: A large colony which is typical for Bt, the surface ofwhich looks dark and dull.

Cell morphology: A typical one for Bt.

Serotype of H-antigen: 3a3b3c, kurstaki.

Intracellular component: A sporulating cell produces a diamond-shapedcrystal which sometimes contains a small cuboidal crystal.

Plasmid: A pattern of a plasmid of the strain on an agaroseelectrophoresis is clearly distinguished from that of the known strainssuch as HD-1, HD-73 and HD-263. Alkaline-soluble protein: The strain hasthe proteins which run to about 130,000 dalton and about 60,000 daltonon the electrophoresis.

Activity: The strain has an insecticidal activity which is from 2 to 50times higher than the known HD-1 and commercially available Bt agentsagainst all the lepidopterous insect pests tested. The strain also showsa stronger insecticidal activity than HD-1 against dipterous insectpest, mosquito. However, it has a lower insecticidal activity than theknown HD-1 and the commercially available Bt agents against a beneficiallepidoptera, Bombyx mori.

These three strains produce the toxic proteins which are different fromthat of HD-1 in a digestion pattern by proteases and various digestivejuices of insects. For example, 130 Kd protein of the toxic proteinderived from HD-1 will be digested into a smaller molecule by trypsin.On the other hand, the corresponding proteins from the strains of thepresent invention are hardly digested by trypsin, and even if digested,a pattern on the electrophoresis of the resulting molecules is differentfrom that of HD-1 (FIG. 1).

As seen from the above data, although the three novel strains aresimilar to the known HD-1 in view of morphological features, they aredistinguished from the known strain, HD-1.

The strains, No.145, No.116 and No.161 can be cultured in a standardfermentation method and medium.

The medium may contain as carbon source sucrose, maltose, glucose,fructose, molasses, soluble starch and the like.

As nitrogen source, there may be mentioned ammonium sulfate, ammoniumchloride, cottonseed powder, yeast extract, soybean cake, caseinhydrolysate and the like.

Minerals and vitamins may be supplied by the above carbon or nitrogensources, which may, however, be added separately.

The strains of the present invention are cultured preferably at pH offrom 5 to 8 and a temperature of from 25° to 30° C. for 2 and 5 days.The culture is carried out preferably in a stirred and aerated system, asubmerged culture in the same system being preferred in amass-production.

The crystal toxic protein may be isolated and collected from a culturemedium by a conventional manner such as centrifugation and filtration.Alternatively, the culture medium containing the strain per se and/orfree crystal toxic protein may be concentrated or dried to give a powderby a conventional method such as a spray-drying, as long as the methoddoes not deteriorate the toxic activity of the protein.

In case the resulting crystal toxin comprises viable cells, such cellswill be sterilized in a conventional method such as heat treatment,ultrasonication, homogenation, physical treatment such as radiation,chemical treatment such as those with formalin, hydrogen peroxide,sulfite salts, chlorine compounds, β-propiolactone, surfactants,ethylene oxide, and propylene oxide and biological treatment such asautolysis, phage treatment and lysozyme treatment. From an industrialpoint of view, heat treatment, homogenation and chemical treatment arepreferred. It is also noted that the treatment should not deterioratethe activity of the crystal toxic protein.

When the crystal toxin component according to the present invention isused in an insect pest-controlling agent, it may be mixed with a naturaland mineral fiber such as talc and kaolin, a solid carrier such as apumice stone, bentonite and diatomite, and a liquid carrier such aswater, and optionally supplemented with an emulsifier, dispersant,suspensions, penetrating agent, spreader and stabilizer. The agent maybe formulated for a practical use into any form such as a wettablepowder, powder, granule, flowable agent.

The agent according to the present invention may be formulated orsprayed together with other agents such as herbicide, pesticide,fungicide, plant-growth regulator, synergist, attractant, plant-nutrientand fertilizer.

An application amount of the crystal toxin-containing material (activecomponent) may be varied depending on an application place, time andmethod, pest insects to be treated and crop to be protected, the activecomponent usually being 0.1-99%, preferably 0.5-50% by weight of theagent.

All parts, percentages and proportions referred to herein and claims areby weight unless otherwise indicated.

Examples of the amounts of each component in the agent of the presentinvention will be summarized as follows:

    ______________________________________                                        active                          (others auxiliary                             component    carriers surfactant                                                                              substance)                                    ______________________________________                                        wettable                                                                             1-70      15-93    3-10    0-5                                         powder                                                                        powder 0.01-30   67-99.5          0-3                                         granule                                                                              0.01-30   67-99.5          0-8                                         flowable                                                                             1-70      10-90    1-20     0-10                                       agent                                                                         ______________________________________                                    

When applied, a wettable powder and flowable agent are diluted with apredetermined amount of water, powder and granular agent may be directlyapplied without dilution.

Each component of the agent may be exemplified as follows:

(Wettable powder)

    ______________________________________                                        active component:                                                                          the crystal toxin-containing material                                         according to the present invention;                              carriers:    calcium carbonate, kaolinite, Sieglite D,                                     Sieglite PEP, diatomite, talc;                                   surfactant:  calcium lignosulfonate, Solpol, Lunox;                           others:      Carplex #80.                                                     ______________________________________                                    

(Powder)

    ______________________________________                                        active component:                                                                          the crystal toxin-containing material                                         according to the present invention;                              carriers:    calcium carbonate, kaolinite, Sieglite D,                                     diatomite, talc;                                                 others:      diisopropyl phosphate, Carplex #80.                              (Granule)                                                                     active component:                                                                          the crystal toxin-containing material                                         according to the present invention;                              carriers:    wheat flour, wheat bran, corn grits,                                          Sieglite D;                                                      others:      paraffin, soybean oil.                                           (Flowable agent)                                                              active component:                                                                          the crystal toxin-containing material                                         according to the present invention;                              carriers:    water;                                                           surfactant:  Solpol, sodium lignosulfonate, Lunox,                                         Nippol;                                                          others:      ethyleneglycol, propyleneglycol.                                 ______________________________________                                    

The insect pest-controlling agent according to the present inventionwill be illustrated below with reference to the following unlimitedformulation examples.

Formulation Example 1

Wettable powder

    ______________________________________                                        the crystal toxin-containing material                                                                     25 parts                                          according to the present invention                                            Sieglite PEP (a trade name of mixture of kaolinite and                                                    66 parts                                          sericite; manufactured by Sieglite Industry Co.)                              Solpol 5039 (a trade name of anionic surfactant;                                                          4 parts                                           manufactured by Toho Chemical Co.)                                            Carplex #80 (a trade name of white carbon; Shionogi                                                       2 parts                                           Pharmaceutical Co.)                                                           ______________________________________                                    

The above components are homogeneously mixed and pulverized to give awettable powder. On application, the wettable powder is diluted 500 to2,000 times and sprayed so that the crystal toxin-containing materialcan be provided at 0.1-5 kg per hectare.

Formulation Example 2

Powder

    ______________________________________                                        the crystal toxin-containing material                                                                  3.0 parts                                            according to the present invention                                            Clay                     95 parts                                             diisopropyl phosphate    1.5 parts                                            Carplex #80              0.5 parts                                            ______________________________________                                    

The above components are homogeneously mixed and pulverized to give apowder. On application, the powder is sprayed so that the crystaltoxin-containing material can be provided at 0.1-5 kg per hectare.

Formulation Example 3

Flowable agent

    ______________________________________                                        the crystal toxin-containing material                                                                    35 parts                                           according to the present invention                                            Lunox 1000C (a trade name of anionic surfactant;                                                         0.5 parts                                          manufactured by Toho Chemical Co.)                                            Solpol 3353 (a trade name of nonionic surfactant;                                                        10 parts                                           manufactured by Toho Chemical Co.)                                            1% Xanthene gum aqueous solution                                                                         20 parts                                           Water                      34.5 parts                                         ______________________________________                                    

The above components except the crystal toxin-containing material arehomogeneously dissolved, mixed with the crystal toxin-containingmaterial, well stirred and wet-pulverized by a sand mill to give aflowable powder. On application, the flowable powder is diluted 50 to2,000 times and sprayed so that the crystal toxin-containing materialcan be provided at 0.1-5 kg per hectare.

It is well known that the toxic protein produced by Bt is encoded by thegene (CP gene) contained in a huge plasmid of the strain. Accordingly,the gene coding for the insecticidal crystal toxin can be cloned,introduced into bacteria such as E. coli and Pseudomonos or targetplants to be protected from lepidopterous insect pests by means of aknown genetic engineering technology so as to exterminate said pests.

An example of the method for the protection of a plant from damagecaused by the lepidopterous insect pests is to treat or spray a plantwith the above agent diluted with, for example, water. The activecomponent of the agent is a toxic δ-endotoxin. The agent according tothe present invention may comprise the toxic δ-endotoxin per se or thestrains producing it. Thus, it is not generally necessary to isolate thetoxic δ-endotoxin from the strains.

Another example of the method is to prepare a plant susceptible to saiddamage so that it can produce in vivo the toxic δ-endotoxin. Thepreparation may be carried out by cloning the genes coding for theδ-endotoxin from the novel strains No.145, No.116 and No.161, ligatingthem with a suitable promoter which can permit an expression of the samegenes in the plant, for example, CaMV35S promoter, transforming theplant in a known manner such as Ti plasmid and electroporation.

The insect pests which can be eradicated by the present method are, forexample, Lepidoptera such as Mamestra brassicae comprising Spodopteralitura and Spodoptera exigua, Plutella xylostella, Cnaphalocrocismedinalis, Chilo suppressalis, Parnara guttara, Pieris rapae crucivora,Monema flavescens, Papilio machaon hippocrates and Diptera such as Aedesaegipti, Culex pipiens pallens, Aedes albopictus and Anopheles hyrcanussinensis.

The present method can protect a wide variety of plants such asvegetables like Brassica oleracea var. capitata, fruit vegetables like acauliflower, citrus fruits, defoliation fruits and a flowering tree aswell as trees in non-crop land such as a plantation, park and forest.

EXAMPLE EXAMPLE 1

Isolation of Bt vat. kurstaki No.145, No.116 and No.161 strains:

One gram of each soil sample obtained in Hokkaido area of Japan (Memuroarea for No. 145, Dounan area for No. 116 and Moshiri area for No. 161)was taken into an Erlenmeyer flask. To the flask was added 10 ml of asterilized distilled water and shaked for 30 min. and then allowed tostand still. A supernatant liquid (2 ml) was collected and heated at 98°C. for 10 min. The resulting solution was diluted ×10 and ×100respectively. Each of the diluted solution (1 ml) was incubated in anusual nutrient agar medium (meat extract 0.3%, peptone 0.3% and agar1.5%, pH 7.0) on a 9 cm-Petri dish at 30° C. for 24-48 hours. Theresulting colony was inoculated in an usual slant medium and incubatedat 30° C. for 4-6 days. After a discriminating staining of an endosporeand crystal, production of the crystal protein was detected by a phasecontrast microscope (×1,500 by means of an oil-immersion lens). Thecrystal protein was usually in a bipyramidal form.

A crystal-positive colony was streak smeared on an usual nutrient agarplate medium and incubated at 30° C. for 3 days to obtain a pure cultureproduct. The discriminating staining was repeated to confirm thepresence of the crystal and to check the purity thereof. The purifiedcolony was transplanted in an agar slant medium and stored.

EXAMPLE 2

Purification of the crystal toxic protein:

The strains according to the present invention were inoculated in theusual nutrient agar medium and incubated at 30° C. After a suitableperiod of time, a part of the incubated strains was collected andchecked about the production of the crystal protein by the phasecontrast microscope (×1,500). Tris-HCl buffer (50 mM) was added to theplate medium to collect the strains, which were then centrifuged torecover a mixture pellet of the strains and crystal. In order to removecontaminants and spores which were further eluted, the following stepswere carried out according to the method described in Protein,Nucleotide and Enzyme; 29, 444-454, 1984; Keiji Yamamoto. The pellet wasmixed with 1M saline and the resulting suspension was shaked hard tofoam. After removal of the foam containing many floating endospores onthe surface of the suspension, the suspension was again centrifuged andthe supernatant was discarded. These serial procedures were repeatedthree times. The resulting pellet was then washed with a steriledistilled water, centrifuged, suspended with a small amount of sterilewater and subjected to Percoll treatment to isolate the crystal toxin(Toshihiko Iizuka, Chemistry and Biology 27, 287-302, 1989).

EXAMPLE 3

Examination of a digestion pattern of the crystal toxic protein bytrypsin:

The crystal toxic protein obtained in EXAMPLE 2 was resolubilized withan alkaline solution, incubated with trypsin at 37° C. for apredetermined period of time and subjected to 10% SDS-polyacrylamide gelelectrophoresis to examine its phoresis pattern. As a control, the knownstrain HD-1 was used after it had been incubated and treated in the samemanner as the strains according to the present invention.

The results are shown in FIG. 1.

EXAMPLE 4

Culture of the strains:

A platinum loopful of the strains of the present invention wasinoculated in 5 ml of the usual nutrient liquid medium in a test tubeand incubated with a reciprocal shaker at 30° C. for 12 to 24 hours toobtain a seed culture. The seed culture was inoculated into anErlenmeyer flask (500 ml) containing 100 ml of Potato-Dextrose medium(potato infusion 20%, dextrose 2%, pH7.0) at a final concentration of 1%and incubated at 30° C. for 50 to 70 hours with a rotary shaker at 200rpm. The cells, spores and crystal protein were recovered bycentrifugation. The resulting pellet was mixed with a certain amount ofwater, subjected to ultrasonication, centrifuged and lyophilized. Afterbeing weighed and adjusted in concentration, the resulting dry productwas subjected to the following insecticidal tests.

EXAMPLE 5

Insecticidal test of the strains on Spodoptera litura:

Leaves of cabbage were impregnated for about 10 sec. with apredetermined amount of the solution of the strains prepared in EXAMPLE4 and supplemented with a spreader, air-dried and taken into a styrolcup containing a wet filter paper. Ten larvae of Spodoptera litura (2larval instars) were released within each of the cup provided with acover having pores and incubated at 25° C. As a control, the knownstrain HD-1 was incubated and treated in the same manner as the strainsaccording to EXAMPLE 4. The test was performed in a two-zone system. Amortality rate at 3 days after the release was calculated by thefollowing equation.

    Mortality rate=(the number of the died insects/the number of the released insects)×100

The results are shown in TABLE 1.

                  TABLE 1                                                         ______________________________________                                        Sample      Conc. (ppm)                                                                              Mortality rate (%)                                     ______________________________________                                        No. 145     500        100                                                                100        100                                                                 10        66.7                                                                 1        9.0                                                    No. 116     500        100                                                                100        75                                                                  10        20                                                                   1        0                                                      No. 161     500        100                                                                100        90                                                                  10        50                                                                   1        43.3                                                   HD-1        500        100                                                                100        25                                                                  10        11.1                                                                 1        0                                                      Control       0        0                                                      ______________________________________                                    

EXAMPLE 6

Insecticidal test of the strains on Plutella xylostella:

Leaves of cabbage were impregnated for about 10 sec. with apredetermined amount of the solution of the strain prepared in EXAMPLE 4and supplemented with a spreader, air-dried and taken into a styrol cupcontaining a wet filter paper. Ten larvae of Plutella xylostella (2larval instars) were released within each of the cup provided with acover having pores and incubated at 25° C. As a control, the knownstrain HD-1 was incubated and treated in the same manner as the strainaccording to EXAMPLE 4. The test was performed in a two-zone system. Amortality rate at 3 days after the release was calculated by thefollowing equation.

    Mortality rate=(the number of the died insects/the number of the released insects)×100

The results are shown in TABLE 2.

                  TABLE 2                                                         ______________________________________                                        Sample      Conc. (ppm)                                                                              Mortality rate (%)                                     ______________________________________                                        No. 145     1          100                                                                0.1        100                                                                0.01       65                                                                 0.001      20                                                     No. 116     1          100                                                                0.1        100                                                                0.01       90                                                                 0.001      80                                                     No. 161     1          100                                                                0.1        100                                                                0.01       66.6                                                               0.001      20                                                     HD-1        1          100                                                                0.1        57.5                                                               0.01       33.3                                                               0.001      0                                                      Control     0          0                                                      ______________________________________                                    

EXAMPLE 7

Insecticidal test of the strains on Spodoptera exigua:

Leaves of cabbage were impregnated for about 10 sec. with apredetermined amount of the solution of the strain prepared in EXAMPLE 4and supplemented with a spreader, air-dried and taken into a styrol cupcontaining a wet filter paper. Ten larvae of Spodoptera exigua (2 larvalinstars) were released within each of the cup provided with a coverhaving pores and incubated at 25° C. As a control, the known strain HD-1was incubated and treated in the same manner as the strain according toEXAMPLE 4. The test was performed in a two-zone system. A mortality rateat 3 days after the release was calculated by the following equation.

    Mortality rate=(the number of the died insects/the number of the released insects)×100

The results are shown in TABLE 3.

                  TABLE 3                                                         ______________________________________                                        Sample      Conc. (ppm)                                                                              Mortality rate (%)                                     ______________________________________                                        No. 145     500        100                                                                100        100                                                                 10        60                                                                   1        18.1                                                   No. 116     500        90                                                                 100        80                                                                  10        33.3                                                                 1        0                                                      No. 161     500        100                                                                100        62.5                                                                10        40                                                                   1        11.1                                                   HD-1        500        100                                                                100        100                                                                 10        11.1                                                                 1        0                                                      Control       0        0                                                      ______________________________________                                    

EXAMPLE 8

Mass-culture of the strains:

A platinum loopful of the strains of the present invention wasinoculated in 10 ml of the usual nutrient liquid medium in an Erlenmeyerflask (100 ml) and incubated with a rotary shaker at 200 rpm at 30° C.for 12 to 24 hours to obtain a seed culture. The seed culture wasinoculated into an Erlenmeyer flask (5000 ml) containing 1000 ml ofPotato-Dextrose medium (potato infusion 20%, dextrose 2%, pH7.0) at afinal concentration of 1% and incubated at 28° C. for 3 to 5 days with arotary shaker at 140 rpm. The cells, spores and crystal protein wererecovered by centrifugation. The resulting pellet was mixed with acertain amount of water, subjected to ultrasonication, centrifuged andlyophilized. After being weighed and adjusted in concentration, theresulting dry product was subjected to the following insecticidal tests.

EXAMPLE 9

Effect of the strains on larvae of Pieris rapae crucivora and Mamestrabrassicae:

The solution (125 ppm) of the strains No.145 and No.161 prepared inEXAMPLE 8 and the solution (140 ppm) of a commercially available BTagent, Toarrow (a registered mark of Toagosei Chemical Industry Co.,Ltd.) were supplemented with a spreader, which were then subjected to afield test on larvae of Pieris rapae crucivora and Mamestra brassicae ina cabbage farm. The tests were carried out in duplicate for 10 strainsper zone.

The results are shown in TABLE 4.

                  TABLE 4                                                         ______________________________________                                        Insect pests (number/10 strains)                                              Pieris rapae crucivora/Mamestra brassicae                                                    After spray                                                    Sample    Before spray                                                                             4 days  6 days                                                                              8 days                                                                              10 days                              ______________________________________                                        No. 145   70/16      11/4    1/1   2/7   0/11                                 No. 161   74/20      6/4     3/20  3/10  2/16                                 Toarrow*   48/140    11/16   9/75  5/72  7/72                                 No spray  42/64      32/43   33/102                                                                              83/110                                                                              51/86                                ______________________________________                                         *Toarrow 7% WP                                                           

EXAMPLE 10

Effect of the strains on larvae of Pieris rapae crucivora and Pluttelaxylostella:

The solution (100 ppm) of the strains No.145, No.116 and No.161 preparedin EXAMPLE 8 and the solution (140 ppm) of a commercially available BTagent, Toarrow (a registered mark of Toagosei Chemical Industry Co.,Ltd. ) were supplemented with a spreader, which were then subjected to afield test on larvae of Pieris rapae crucivora and Pluttela xylostellain a cabbage farm. The tests were carried out in duplicate for 24strains per zone.

The results are shown in TABLE 5.

                  TABLE 5                                                         ______________________________________                                        Insect pests (number/10 strains)                                              Pieris rapae crucivora/Pluttela xylostella                                                       After spray                                                Sample    Before spray   4 days   8 days                                      ______________________________________                                        No. 145   163/96         4/34     17/58                                       No. 116   131/69         5/23     12/42                                       No. 161   147/72         5/48     24/99                                       Toarrow*  179/77         17/42    19/79                                       No spray   174/108       161/331  213/311                                     ______________________________________                                         *Toarrow 7% WP                                                           

EXAMPLE 11

Insecticidal test of the strains on larvae of Bombyx mori:

The solution of the strains prepared in EXAMPLE 4 and Toarrow 7% WP werehomogeneously mixed with 10 gram of an artificial feed. The resultingfeed was taken in 2 to 3 cm height into a styrol cup. Ten larvae ofBombyx mori (3 larval instars) were released within each of the cup andincubated at 25° C. A mortality rate at 5 days after the release wascalculated by the following equation.

    Mortality rate=(the number of the died insects/the number of the released insects)×100

The results are shown in TABLE 6.

                  TABLE 6                                                         ______________________________________                                        Sample        Conc. (ppm)                                                                              Mortality rate (%)                                   ______________________________________                                        No. 145       100        20                                                                 50         0                                                                  25         0                                                                  12.5       0                                                                  6.25       0                                                    No. 116       100        100                                                                50         65                                                                 25         20                                                                 12.5       5                                                                  6.25       0                                                    No. 161       100        100                                                                50         70                                                                 25         20                                                                 12.5       10                                                                 6.25       0                                                    Toarrow 7% WP 100        100                                                                50         100                                                                25         100                                                                12.5       100                                                                6.25       80                                                   Control       0          0                                                    ______________________________________                                    

EXAMPLE 12

Insecticidal test of the strains on Aedes albopictus:

The solution of the strains prepared in EXAMPLE 4 were diluted withsterilized distilled water and taken into a deep laboratory dish. Tenlarvae of Aedes albopictus (2 larval instars) were released within thedish and incubated at 25° C. The tests were carried out in a two-zonesystem. HD-1 and israelensis strains were also subjected to the test. Amortality rate at 3 days after the release was calculated by thefollowing equation.

    Mortality rate=(the number of the died insects/the number of the released insects)×100

The results are shown in TABLE 7.

                  TABLE 7                                                         ______________________________________                                        Sample      Conc. (ppb)                                                                              Mortality rate (%)                                     ______________________________________                                        No. 145     1000       100                                                                100        100                                                                 10         30                                                                  1         20                                                    No. 116     1000       100                                                                100        100                                                                 10        100                                                                  1         20                                                    No. 161     1000       100                                                                100        100                                                                 10         40                                                                  1         40                                                    HD-1        1000        50                                                                100          0                                                                 10          0                                                                  1          0                                                    israelensis 1000       100                                                                100        100                                                                 10        100                                                                  1         30                                                    Control       0          0                                                    ______________________________________                                    

What is claimed is:
 1. An insect pest-controlling agent comprising as anactive component an insecticidal crystal toxin produced by the strainselected from the group consisting of Bacillus thuringiensis var.kurstaki No. 145 FERM BP-3905(Bt. k 145), Bacillus thuringiensis var.kurstaki No. 161 FERM BP-3906, (Bt. k 161), and Bacillus thuringiensisvar. kurstaki No. 116 FERM BP-3907 (Bt. k 116), wherein the insecticidalcrystal toxin has a set of molecular weights as determined by 10%SDS-polyacrylamide gel electrophoresis of about 125,000 daltons andabout 60,000 daltons when produced by Bt. k 145, about 130,000 daltonsand about 60,000 daltons when produced by Bt. k 161, and about 130,000daltons and about 60,000 daltons when produced by Bt. k
 116. 2. Theinsect pest-controlling agent according to claim 1, which comprises asolid diluent, a surfactant, or a combination of a solid diluent and asurfactant.
 3. A method for protection of a plant from damage caused byan insect pest selected from the group of insects consisting of theorder Lepidoptera and Diptera, comprising feeding the insect pest withthe insecticidal crystal toxin produced by the strain Bacillusthuringiensis var. kurstaki No. 145 FERM BP-3905 (Bt. k 145), Bacillusthuringiensis var. kurstaki No. 161 FERM BP-3906, (Bt. k 161), andBacillus thuringiensis var. kurstaki No. 116 FERM BP-3907 (Bt. k 116),wherein the insecticidal crystal toxin has a set of molecular weights asdetermined by 10% SDS-polyacrylamide gel electrophoresis of about125,000 daltons and about 60,000 daltons when produced by Bt. k 145,about 130,000 daltons and about 60,000 daltons when produced by Bt. k161, and about 130,000 daltons and about 60,000 daltons when produced byBt. k
 116. 4. A method according to claim 3, comprising treating a plantwith an insecticidally effective amount of the crystal toxin prior to orduring infestation from the insect pest.
 5. A method according to claim3, wherein the insect pest is from the order Lepidoptera and is selectedfrom the group consisting of Mamestra brassicae, Plutella xylostella,Chaphalocrocis medinalis, Chilo suppressalis, Parnara guttata, Pierisrapae crucivora, Monema flavescens, Spodoptera litura, Spodopteraexigua, and Papilio machaon hippocrates.
 6. A method according to claim3, wherein the insect pest is from the order Diptera and is selectedfrom the group consisting of Aedes aegipti, Culex pipiens palleus, Aedesalbopictus and Anopheles hycrcanus sinensis.
 7. The insect controllingagent of claim 1, wherein the insecticidal crystal toxin produced is adiamond shaped crystal, which when impregnated on cabbage leaves forabout 10 seconds at a concentration of 10 ppm against Spodoptera lituraincubated at 25° C. has a mortality of about 66% for Bt. k. 145, about20% for Bt. k 116 and about 50% for Bt. k
 161. 8. The method accordingto claim 3, wherein the insecticidal crystal toxin produced is a diamondshaped crystal, which when impregnated on cabbage leaves for about 10seconds at a concentration of 10 ppm against Spodoptera litura incubatedat 25° C. has a mortality of about 66% for Bt. k. 145, about 20% for Bt.k 116 and about 50% for Bt. k
 161. 9. The insect pest-controlling agentof claim 2, wherein the surfactant is an anionic or nonanionicsurfactant.